Culture device and culture method for preparing separated assembloid

Abstract

A culture device for preparing separated assembloid comprises a first culture chamber, a second culture chamber and at least one assembloid culture unit, wherein the assembloid culture unit at least comprises a first cell culture cavity, a second cell culture cavity and one or more directional channels, wherein the first cell culture cavity and the second cell culture cavity are configured to culture a first cell and a second cell, respectively; the directional channels are filled with a directional induction growth culture medium to allow the first cell and / or the second cell to undergo directional axon-induced growth in the directional induction growth culture medium; and the first culture cavity and the second culture cavity are respectively configured to contain a first culture medium suitable for the growth of the first cell and a second culture medium suitable for the growth of the second cell, neither of which is mixed with the other. The assembloid built by the culture device can not only be used for basic research on the growth, projection, and synaptogenesis of neuron axons from a human source, but also provides a research model for human neurological diseases, and neuronal axon regeneration and myelination.

Description

Culture device and method for preparing separated combined organoids Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to a culture device for preparing separated combined organoids, and a culture method for separated combined organoids. Background Art

[0002] Organoids are in vitro cultures derived through 3D induction methods that mimic the cellular composition and structure of in vivo organs. Compared to 2D cells, organoids are more similar to in vivo tissues. Human organoids, in particular, bridge the gap between species differences in model animals and have significant application value. However, brain organoids are currently only available for single brain regions, precluding studies of inter-regional projections and function.

[0003] Assembloid is an emerging concept in organoid culture, which involves co-culturing different types of organoids, and can be used to study the interactions between different organoids. For example, thalamic organoids and telencephalic organoids are co-cultured to study the projections and functions between the thalamus and telencephalon. Assembloids in existing technologies all adopt a fusion approach, in which two or more organoids are directly contacted and cultured with the same culture medium. A major disadvantage of culturing assembloids by direct fusion is that different types of organoids must be cultured in the same culture medium, which is not conducive to the survival, differentiated differentiation, and maturation of organoids.

[0004] Summary of the Invention

[0005] This application discloses a culture device for preparing separated combined organoids, as well as a culture method for separated combined organoids, which enables any homologous or heterologous organoids to form separated combined organoids through long-range axonal connections, and heterologous organoids can grow in their respective most suitable culture media.

[0006] The present application discloses a culture device for preparing separated combined organoids, the culture device comprising:

[0007] at least one combined organoid culture unit and its corresponding culture chamber;

[0008] Wherein, the combined organoid culture unit comprises at least a first cell culture chamber, a second cell culture chamber, and one or more directional channels for connecting the cell culture chambers in the combined organoid culture unit;

[0009] Wherein, the first cell culture chamber is configured to culture first cells, and the first cells are human or animal cells;

[0010] The second cell culture chamber is configured to culture second cells, wherein the second cells are human or animal cells;

[0011] The directional channel is filled with a directional growth induction culture medium, thereby allowing the first cells and / or the second cells to produce directional axon-induced growth in the directional growth induction culture medium;

[0012] The first cell culture chamber is located in the first culture chamber, and the second cell culture chamber is located in the second culture chamber; the first culture chamber is configured to contain a first culture medium suitable for the growth of first cells, and the second culture chamber is configured to contain a second culture medium suitable for the growth of second cells; and the first culture medium and the second culture medium are configured not to mix with each other.

[0013] In a preferred embodiment, the first cell and the second cell are the same or different.

[0014] In a preferred embodiment, the first cell is selected from any one of the following:

[0015] Neurons, brain organoids, retinal organoids, spinal cord organoids; or

[0016] Glial cells, muscle organoids, heart, pancreas, small intestine and other visceral organoids, tumor organoids.

[0017] In a preferred embodiment, the second cell is selected from the group consisting of neurons, brain organoids, retinal organoids, and spinal cord organoids; or

[0018] Glial cells, muscle organoids, cardiac organoids, pancreatic organoids, small intestinal organoids, tumor organoids.

[0019] In a preferred embodiment, the directional growth induction culture medium is an axon directional growth induction culture medium.

[0020] In a preferred embodiment, the directed growth induction culture medium is Matrigel.

[0021] In a preferred embodiment, the cells are derived from any one of the following: mammals, rodents, non-human primates, and humans.

[0022] In a preferred embodiment, after culturing, the first cell culture formed by the first cells and the second cell culture formed by the second cells form a combined state in the directional channel, thereby forming a separated combined organoid;

[0023] In another preferred embodiment, the first cells form a first cell culture, and / or the second cells form a second cell culture; wherein,

[0024] The first cell culture and / or the second cell culture is an organoid.

[0025] In a second aspect, the present application discloses a method for culturing isolated composite organoids, wherein the method is configured to construct composite organoids in an isolated manner; the culturing method comprises:

[0026] - Using a culture device to achieve directional growth of axons in three-dimensional space by occupying the channels of the culture device and applying Matrigel;

[0027] - Place isolated homologous or xenogeneic organoids in different chambers on both sides;

[0028] -The organoids in different chambers on both sides are connected by axons to form combined organoids;

[0029] In a preferred embodiment, when the organoids in the two side chambers need to be grown in different culture media, a baffle is set in the middle of the two side chambers to separate the two side chambers, so that the heterogeneous organoids can be grown in their respective most suitable culture media.

[0030] In another preferred embodiment, the culture device is configured with a plurality of different chambers and the chambers are connected by pipes.

[0031] In a third aspect, the present application further discloses a method for culturing separate combined organoids, wherein the method cultures neural cells in multiple culture areas, and the neural cells in the culture areas perform neural projection between the multiple culture areas.

[0032] In a preferred embodiment, the plurality of culture areas are configured with corresponding culture media according to the neural cells in the culture areas, and the culture media are not mixed with each other.

[0033] In a preferred embodiment, directional channels are arranged between the culture areas, and a directional induction growth culture medium is arranged in the directional channels. The nerve cells produce directional axon induced growth through the directional induction growth culture medium, thereby forming neural projections.

[0034] In a preferred embodiment, the directed growth induction culture medium is Matrigel.

[0035] In a fourth aspect, the present application further discloses a separate combined organ, which includes a plurality of nerve aggregation regions, wherein projections are formed between the plurality of nerve aggregation regions through the directional growth of axons.

[0036] In a preferred embodiment, the separated combined organoids are human retinal organoids and the human retinal organoids form projections through three-dimensionally directed long-range axons and thalamic organoids.

[0037] In a fourth aspect, the present application further discloses an axon regeneration model, which is constructed using the culture device as described above.

[0038] In a fifth aspect, the present application further discloses an axon myelination model, which is constructed using the culture device as described above.

[0039] This application has at least the following beneficial technical effects:

[0040] 1) Organoids differentiated from patient-derived induced pluripotent stem cells are used to build composite organoids on this device to construct disease models for the study of diseases related to axonal growth, projection, and synapse formation.

[0041] 2) Unlike fused organoids, where neuronal axons grow internally, organoids constructed using this device are isolated, with axons growing within the organoid's connecting tubes. Examples include projections between the optic nerve and the thalamus, between different brain regions, and between motor neurons and muscles.

[0042] 3) The device described in this application, as a structural unit, not only enables unidirectional and bidirectional projection, but can also be expanded to one-to-many, many-to-one, and multi-stage combination culture of three or more organoids. In addition to neuronal projection, this device can also theoretically be used to induce the growth of vascular organoids in a directional manner.

[0043] The specification of this application records a large number of technical features, which are distributed in various technical solutions. If all possible combinations of technical features of this application (i.e., technical solutions) are to be listed, the specification will be too lengthy. In order to avoid this problem, the various technical features disclosed in the above-mentioned invention content of this application, the various technical features disclosed in the various embodiments and examples below, and the various technical features disclosed in the accompanying drawings can be freely combined with each other to form various new technical solutions (these technical solutions should all be deemed to have been recorded in this specification), unless such a combination of technical features is technically infeasible. For example, in one example, feature A+B+C is disclosed, and in another example, feature A+B+D+E is disclosed. Features C and D are equivalent technical means that play the same role. Technically, only one of them can be used, and it is impossible to use them at the same time. Feature E can be technically combined with feature C. Then, the solution of A+B+C+D should not be deemed to have been recorded because it is technically infeasible, while the solution of A+B+C+E should be deemed to have been recorded. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following is a brief introduction to the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0045] FIG1 is a model diagram and a physical diagram of a mold for making the culture device of the present application;

[0046] FIG2 is a physical diagram of the culture device of the present application prepared using a mold and agarose;

[0047] FIG3 is a schematic diagram of the implantation and culture of a first organoid according to the culture method of the present application;

[0048] FIG4 is a schematic diagram of axons of a first organoid sample obtained according to the culture method of the present application, growing in a 3D directional manner in a tube;

[0049] FIG5 is a schematic diagram of the implantation and culture of a second organoid according to the culture method of the present application;

[0050] FIG6 is a schematic diagram of isolated retinal-thalamic combined organoids connected by long-range axons obtained according to the culture method of the present application;

[0051] FIG7 is a schematic diagram showing the characterization of synapses formed in the retinal-thalamic combined organoids obtained according to the culture method of the present application;

[0052] FIG8 is a schematic diagram of a human optic nerve regeneration model constructed according to the culture device and culture method of the present application. DETAILED DESCRIPTION

[0053] After extensive research, the inventors of this application have proposed a separate composite organoid culture device and culture method. By leveraging the long-range projection properties of axons of certain types of neurons (such as retinal ganglion cells and cross-brain projection neurons), the separate composite organoid culture device not only enables 3D directional growth of neuronal axons, but also allows different types of organoids to differentiate and mature in their respective optimal culture media, with axons projecting and connecting. Composite organoids cultured using this culture device can be used to study the mechanisms of axon growth, projection, and synapse formation in various types of human neurons, and to screen for regulatory factors that influence neuronal projection and synapse formation. Furthermore, by using organoids differentiated from patient-derived induced pluripotent stem cells, composite organoids can be constructed on this device to create disease models, allowing the study of diseases related to axon growth, projection, and synapse formation. Furthermore, unlike fused composite organoids, where neuronal axons are internal to the organoid, composite organoids constructed using this device are separate, with axons growing within the tubes connecting the organoids. Therefore, it is very convenient to study the regeneration and myelination of neuronal axons and explore the conditions for axon regeneration and myelination of various types of human neurons. In summary, the combined organoids constructed using this device can not only conduct basic research on the axon growth, projection and synapse formation of various types of human neurons, but also provide a research model for human neurological diseases and neuronal axon regeneration, and have potential clinical application value.

[0054] In the following description, many technical details are provided to help readers better understand this application. However, those skilled in the art will understand that even without these technical details and various changes and modifications based on the following embodiments, the technical solutions claimed in this application can be implemented.

[0055] the term

[0056] As used herein, "separate combined organoid culture device," "combined organoid culture device," "culture device," and "device" are used interchangeably to refer to the culture device of the present application.

[0057] The following is a summary of some of the innovative features of the embodiments of this application:

[0058] The culture device of this application is used to induce 3D directional growth of neuronal axons in organoids and form projections with another organoid, thereby constructing separate combined organoids. The organoids connected by axons can be homologous or heterologous, and heterologous organoids can be cultured in their respective most suitable culture media.

[0059] The culture device includes two parts: the device body and the baffle. The device body is provided with a plurality of independent, open chambers, which are used to accommodate organoids. Connecting pipes are provided between the chambers. These pipes, combined with the physical spacers and the application of Matrigel, realize the 3D directional induced growth of neuronal axons in organoids. The pipes between different chambers can form a one-to-one connection, and / or a one-to-many connection, and / or a many-to-many connection. The baffle is used to separate the chambers on both sides, blocking the connection of the culture medium on both sides of the culture vessel, and realizing the differentiated culture of the organoids on both sides.

[0060] Taking Figure 2 as an example, an embodiment of the culture device of the present application is illustrated: the culture device includes two chambers, a pipe connecting the two chambers, and a baffle set up in the middle of the two chambers. Different types of organoids are placed in the chambers on both sides for separate culture, and Matrigel is injected into the middle pipe connecting the two chambers as a matrix to induce axon growth. The culture media on both sides are separated by a baffle. The organoid grows axons in the chamber on one side and grows in a 3D form in the pipe toward the chamber on the other side, and finally forms a projection with the organoid on the other side. In the embodiments of the present application, retinal organoids and thalamic organoids are used to demonstrate the device. Any different organoids connected by long-range axons can be used for combined organoid culture using this device, and it can be expanded to more than 3 types of organoids for combined culture.

[0061] The separated combined organoid culture device of the present application includes a device body and a baffle. The device body includes a groove-shaped culture unit. In a favorable embodiment, there are multiple culture units on the device body. A first chamber and a second chamber are provided in the direction in which the culture unit extends. The first chamber and the second chamber are in a concave spherical shape with a closed shape at the opening. In an optional embodiment, the diameter of the first chamber is 1.5 mm and the diameter of the second chamber is 1.8 mm.

[0062] The middle pipe between the first chamber and the second chamber connects the two chambers. The middle pipe is used for the 3D directional growth of the axons of the organoid. There are pipe segments on the side of the first chamber away from the middle pipe (i.e. the left side of the first chamber) and on the side of the second chamber away from the middle pipe (i.e. the right side of the second chamber). On the one hand, the pipe segments on both sides provide support for the steel balls to occupy the space, and on the other hand, after the steel balls are removed, the culture medium enters the pipe to provide nutrition and oxygen supply to the organoid. The dimensional parameters such as the length and cross-sectional diameter of the pipe are designed differently according to the cultured combined organoids. In an optional embodiment, the diameter of the pipe on the left side of the first chamber is 0.7mm, the diameter of the middle pipe is 1.0mm, and the diameter of the pipe on the right side of the second chamber is 1.0mm. In the embodiment of the present application, it is preferably designed to adapt to 3.5cm culture dishes, and in theory it can be designed to adapt to any container.

[0063] The baffle is separate from the device body. When in use, the baffle can be mounted on the central axis of the culture dish, with the two ends of the baffle having a shape that is nested and adapted to the device body. The baffle is configured to block fluid communication between different culture media.

[0064] When culturing combined organoids using the culture device of the present application, the types of the first organoid and the second organoid can be the same or different. The organoid can be a combination of any human organoid that can form long-range projection neurons and other types of organoids. The types of organoids that can be cultured in the embodiments include, but are not limited to: various types of neurons, neural tissue organoids, such as retinal organoids, brain organoids, spinal cord organoids, etc.; they can also be cell or tissue organoids that interact with the nervous system, such as glial cells, muscle organoids, visceral organoids such as the heart, pancreas, small intestine, and tumor organoids.

[0065] Mold design of culture device

[0066] Figure 1 shows SOLIDWORKS modeling (Figures A, B, and C) and 3D-printed images (Figures D, E, and F). Figures A and B show the top and bottom views, respectively, of the mold used to form the device body. Figure C shows the 3D-printed baffle. In an alternative embodiment, the mold used to form the device body is made of a resin; in an alternative embodiment, the baffle is made of polycarbonate (PC).

[0067] Fabrication of culture device

[0068] After the mold is prepared, the method for preparing the culture device of the present application using the mold is shown in FIG2 , and includes the following steps:

[0069] 1. Dissolve low-melting-point agarose in DPBS over warm water to create a 3% agarose solution. Sterilize by filtering through a 0.22 μm filter. Agarose is not only biocompatible but also soft, making it easier to remove the organoids later.

[0070] 2. Pour 1.7 ml of 3% agarose solution into a 3.5 cm culture dish and press the mold into the agarose (as shown in Figure 2A).

[0071] 3. After the agarose solution has completely cooled, remove the mold and create the desired chambers and tubes in the agarose (as shown in Figure 2B). Figure 2B and Figure 2C show the back of the culture device and the front of the chambers and tubes under a stereomicroscope, respectively. To prevent the filler in the tubes and the organoids in the chambers from swaying in the culture medium and to keep them fixed, the tubes and chambers are designed with closed ends (Figure 2D and Figure 2E).

[0072] In order to better understand the technical solution of the present application, a specific embodiment is provided below for illustration. The details listed in this example are mainly for ease of understanding and are not intended to limit the scope of protection of the present application.

[0073] Example 1: Implantation and culture of the first organoid

[0074] 1. Insert a sterile steel ball into the left channel of the first chamber. This ball prevents the Matrigel from flowing out of this channel and prevents the left side of the organoid from being wrapped by Matrigel (View A in Figure 3). Simultaneously, insert a sterile steel ball into the right end of the central channel between the two chambers, near the second chamber, to prevent the Matrigel from flowing out of the central channel into the second chamber (View A in Figure 3).

[0075] 2. Use a Pasteur pipette to remove the organoid (here, retinal organoids are used as an example) and place it into the first chamber. Completely aspirate the culture medium from the chamber and tube. The left side of the retinal organoid should now be completely attached to the steel ball in the left tube (Figure 3, View B, indicated by the white arrow).

[0076] 3. Inject Matrigel into the first chamber where the organoid is located and into the middle channel to the right of it (View C in Figure 3), and place the culture device body in a cell culture incubator for 15 minutes to allow the Matrigel to solidify (View D in Figure 3).

[0077] 4. Remove the steel balls on both sides (view E in Figure 3) and add culture medium for cultivation.

[0078] Example 2: 3D Directed Growth of Organoid Axons

[0079] After a period of culture, the retinal organoid samples obtained according to the method of Example 1 showed that the axons of the retinal organoids in the first chamber were located in the middle channel filled with Matrigel (as shown in Figure 4, Panel A) (other growth factors and other substances can be added). On the 20th day, they reached the right end of the channel and were 5.8 mm in length (as shown in Figure 4, Panel B).

[0080] Example 3: Implantation of a Second Organoid

[0081] When the axons of the organoid in the first chamber have grown to a certain length, the second organoid is implanted. A steel ball is embedded in the right channel of the second chamber (view A in Figure 5) to prevent Matrigel from flowing out of the chamber.

[0082] A second organoid (here, a thalamic organoid) was aspirated using a Pasteur pipette and implanted into the second chamber (Figure 5, View B). After the culture medium was aspirated (Figure 5, View C), the chamber was filled with Matrigel (Figure 5, View D). The culture apparatus was placed in an incubator for 15 minutes to allow the Matrigel to solidify. The steel ball in the right channel of the second chamber was removed (Figure 5, View E).

[0083] Example 4: Baffle Installation and Projection Formation

[0084] A PC baffle was placed between the two chambers, and the interface between the baffle and Matrigel was sealed with 3% agarose solution (Figure 6, View A, the baffle is indicated by the white arrow, and 3% agarose was sealed on both sides of the baffle, as indicated by the orange arrow). The purpose of the baffle is to isolate different types of culture medium liquids.

[0085] 1 ml of retinal organoid culture medium was added to the left side of the baffle, and 1 ml of thalamic organoid culture medium was added to the right side of the baffle for subsequent culture (Figure 6 View B). After a period of time, the axons derived from the retinal organoids (expressing EGFP) crossed the tube and grew into the thalamic organoids (Figure 6 View C), and dense axons derived from retinal organoids were visible in the thalamic organoids (Figure 7 View D). The expression of presynaptic protein Synaptophysin (SYP) and postsynaptic protein postsynaptic density protein 95 (PSD95) was detected in the thalamus by immunostaining. Figure 7 View E and Figure 7 View F show the expression of presynaptic protein SYP in thalamic organoids and the expression of postsynaptic protein PSD95 in thalamic organoids, respectively. The vast majority of SYP and PSD95 signals co-localize, and in some areas, SYP, PSD95 and EGFP signals are triple co-localized (Figure 7 View G, white arrows indicate the area where the signals overlap three times).

[0086] The results showed that axons derived from retinal organoids grew into thalamic organoids and formed synapses, completing the projection.

[0087] Example 5: Constructing a human optic nerve regeneration model using this culture device

[0088] As mentioned above, the 3D-oriented growth of neuronal axons is exposed in the tube, making it easy to cut axons and directly observe axon regeneration. The following demonstrates the construction of a human optic nerve regeneration model using retinal organoids as an example.

[0089] Retinal organoids were planted in the first chamber and filled with Matrigel in the middle channel. After a period of time, the channel was filled with axons growing to the right, similar to the human optic nerve, and the retinal organoid axons grew in the channel (Figure 8 View A). The axons were cut using a special blade (Figure 8 View B, white arrows indicate the cut site), and new axons were visible after a few days (Figure 8 View C, new axons, white arrows indicate the cut site, blue arrows indicate new axons).

[0090] Experiments have shown that this device can easily build various neuron axon regeneration models.

[0091] It should be noted that in this patent application, relational terms such as first and second, etc., are used solely to distinguish one entity or operation from another, and do not necessarily require or imply any actual relationship or order between these entities or operations. Furthermore, the terms "comprise," "include," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. Without further limitation, an element specified by the phrase "comprising a" does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising the element. In this patent application, reference to performing an action in accordance with an element means performing the action in accordance with at least that element, including two situations: performing the action in accordance with that element alone, and performing the action in accordance with that element and other elements. Expressions such as "plurality," "multiple times," and "many" include "two," "twice," "two kinds," and "more than two," "more than two times," and "more than two kinds."

[0092] This specification includes combinations of the various embodiments described herein. Separate references to "one embodiment" or a particular embodiment, etc., do not necessarily refer to the same embodiment; however, these embodiments are not mutually exclusive unless indicated as such or clear to one skilled in the art. It should be noted that the word "or" is used in this specification in a non-exclusive sense unless the context clearly indicates or requires otherwise.

[0093] All documents mentioned in this application are considered to be included in their entirety in the disclosure of this application so that they can be used as a basis for modification when necessary. In addition, it should be understood that after reading the above disclosure of this application, those skilled in the art may make various changes or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A culture device for preparing separate combined organoids, characterized in that: The culture device comprises: at least one combined organoid culture unit and its corresponding culture chamber; Wherein, the combined organoid culture unit comprises at least a first cell culture chamber, a second cell culture chamber, and one or more directional channels for connecting the cell culture chambers in the combined organoid culture unit; Wherein, the first cell culture chamber is configured to culture a first cell, and the first cell is a human or animal cell; The second cell culture chamber is configured to culture second cells, wherein the second cells are human or animal cells; The directional channel is filled with a directional growth induction culture medium, thereby allowing the first cell and / or the second cell to produce directional axon induced growth in the directional growth induction culture medium; The first cell culture chamber is located in the first culture chamber, and the second cell culture chamber is located in the second culture chamber; the first culture chamber is configured to contain a first culture medium suitable for the growth of first cells, and the second culture chamber is configured to contain a second culture medium suitable for the growth of second cells; and the first culture medium and the second culture medium are configured not to mix with each other.

2. The culture device according to claim 1, characterized in that The first cell and the second cell are the same or different.

3. The culture device according to claim 1 or 2, characterized in that: The first cell and the second cell are different.

4. The culture device according to claim 1, characterized in that: The first cell is selected from any one of the following: Neurons, brain organoids, retinal organoids, spinal cord organoids; or Glial cells, muscle organoids, heart, pancreas, small intestine and other visceral organoids, tumor organoids.

5. The culture device according to claim 1, characterized in that: The second cell is selected from the group consisting of neurons, brain organoids, retinal organoids, and spinal cord organoids; or Glial cells, muscle organoids, cardiac organoids, pancreatic organoids, intestinal organoids, tumor organoids.

6. The culture device according to claim 1, characterized in that: The directional induced growth culture medium is an axon directional induced growth culture medium.

7. The culture device according to claim 1 or 6, characterized in that: The directional induction growth culture medium is Matrigel.

8. The culture device according to claim 1, characterized in that: The cell origin is any one of the following: mammals, rodents, non-human primates, and humans.

9. The culture device according to claim 1, characterized in that: After culturing, a first cell culture formed by the first cells and a second cell culture formed by the second cells form a combined state in the directional channel, thereby forming a separated combined organoid; In another preferred embodiment, the first cells form a first cell culture, and / or the second cells form a second cell culture; wherein, The first cell culture and / or the second cell culture is an organoid.

10. A method for culturing separated combined organoids, characterized in that: The method is configured to construct a combined organoid in a separate method; the culture method comprises: - Using a culture device, by occupying the channels of the culture device and applying Matrigel, the directional growth of axons in three-dimensional space is achieved.

11. The culture method according to claim 10, characterized in that: The method specifically comprises the following steps: S1: Place isolated homologous or heterologous organoids in different chambers on both sides; S2: The organoids in different chambers on both sides are connected by axons to form combined organoids.

12. The culture method according to claim 11, characterized in that The method also The following steps are involved: S3: When the organoids in the two side chambers need to be grown in different culture media, a baffle is set in the middle of the two side chambers to separate the two side chambers, so that the heterogeneous organoids can grow in their respective most suitable culture media.

13. The culture method according to claim 10, characterized in that: The culture device comprises a plurality of chambers, and the chambers are connected by pipelines.

14. The culture method according to claim 10, characterized in that: The culture device includes a plurality of culture areas, and the culture areas are communicatively connected to each other.

15. A method for culturing separated combined organoids, characterized in that: The culture method cultures neural cells in a plurality of culture regions, and the neural cells in the culture regions perform neural projection between the plurality of culture regions.

16. The culture method according to claim 15, characterized in that: The plurality of culture areas are configured with corresponding culture media according to the nerve cells in the culture areas, and the culture media are not mixed with each other.

17. The culture method according to claim 15, characterized in that: A directional channel is arranged between the culture areas, and a directional induction growth culture medium is arranged in the directional channel. The nerve cells produce directional axon induced growth through the directional induction growth culture medium, thereby forming nerve projection.

18. The culture method according to claim 17, characterized in that: The directed induction growth culture medium is Matrigel.

19. A separated composite organoid, characterized in that: The separated combined organ includes a plurality of nerve aggregation regions, and projections are formed between the plurality of nerve aggregation regions through the directional growth of axons.

20. The split combined organ according to claim 19, characterized in that: The separated combined organoids are human retinal organoids and the human retinal organoids form projections through three-dimensional oriented growth of long-range axons and thalamic organoids.

21. An axon regeneration model, characterized in that: The method is constructed using the culture device according to any one of claims 1 to 9.

22. An axon myelination model, characterized in that: The method is constructed using the culture device according to any one of claims 1 to 9.

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